Synthesis, antitumor activity and mechanism of action of novel 1,3-thiazole derivatives containing hydrazide–hydrazone and carboxamide moiety

Article abstract of DOI:10.1016/j.bmcl.2016.05.059

A series of novel 2,4,5-trisubstituted 1,3-thiazole derivatives containing hydrazide–hydrazine, and carboxamide moiety including 46 compounds T were synthesized, and evaluated for their antitumor activity in vitro against a panel of five human cancer cell lines. Eighteen title compounds T displayed higher inhibitory activity than that of 5-Fu against MCF-7, HepG2, BGC-823, Hela, and A549 cell lines. Especially, T1, T26 and T38 exhibit best cytotoxic activity with IC₅₀values of 2.21?μg/mL, 1.67?μg/mL and 1.11?μg/mL, against MCF-7, BCG-823, and HepG2 cell lines, respectively. These results suggested that the combination of 1,3-thiazole, hydrazide–hydrazone, and carboxamide moiety was much favorable to cytotoxicity activity. Furthermore, the flow cytometry analysis revealed that compounds T1 and T38 could induce apoptosis in HepG2 cells, and it was confirmed T38 led the induction of cell apoptosis by S cell-cycle arrest.

Full text of DOI:10.1016/j.bmcl.2016.05.059

Accepted Manuscript
Synthesis, antitumor activity and mechanism of action of novel 1,3-thiazole de-
rivatives containing hydrazide-hydrazone and carboxamide moiety
Haifeng He, Xiaoyan Wang, Liqiao Shi, Wenyan Yin, Ziwen Yang, Hongwu
He, Ying Liang
PII:
S0960-894X(16)30557-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.059
BMCL 23918
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 January 2016
9 May 2016
21 May 2016
Please cite this article as: He, H., Wang, X., Shi, L., Yin, W., Yang, Z., He, H., Liang, Y., Synthesis, antitumor
activity and mechanism of action of novel 1,3-thiazole derivatives containing hydrazide-hydrazone and carboxamide
moiety, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.059
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Synthesis, antitumor activity and mechanism of action of novel 1,3-thiazole
derivatives containing hydrazide-hydrazone and carboxamide moiety
Haifeng He,a Xiaoyan Wang,a Liqiao Shi,b Wenyan Yinb, Ziwen Yangb, Hongwu He,a,* and Ying
Liangb,*
aKey Laboratory of Pesticide and Chemical Biology of Ministry of Education; College of
Chemistry, Central China Normal University, Wuhan 430079, China.
bNational Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences,
Wuhan 430064, People’s Republic of China
*To whom correspondence should be addressed. E-mail: he1208@mail.ccnu.edu.cn;
liangying2004123@163.com
Abstract: A series of novel 2,4,5-trisubstituted 1,3-thiazole derivatives containing hydrazide-
hydrazine, and carboxamide moiety including 46 compounds T were synthesized, and evaluated
for their antitumor activity in vitro against a panel of five human cancer cell lines. Eighteen title
compounds T displayed higher inhibitory activity than that of 5-Fu against MCF-7, HepG2,
BGC-823, Hela, and A549 cell lines. Especially, T1, T26 and T38 exhibit best cytotoxic activity
with IC50 values of 2.21 μg/mL, 1.67 μg/mL and 1.11 μg/mL, against MCF-7, BCG-823, and
HepG2 cell lines, respectively. These results suggested that the combination of 1,3-thiazole,
hydrazide-hydrazone, and carboxamide moiety was much favorable to cytotoxicity activity.
Furthermore, the flow cytometry analysis revealed that compounds T1 and T38 could induce
apoptosis in HepG2 cells, and it was confirmed T38 led the induction of cell apoptosis by S
cell-cycle arrest.
Keywords: 1, 3-thiazole, carboxamide, hydrazide-hydrazone, antitumor activity, apoptosis.

1,3-thiazole scaffold have attracted considerable attentions for decades due to their remarkable
spectrum of biological activity, such as distinctive antifungal,1,2 antimicrobial,3-6 antitubercular7,8
activity. Moreover, several 1,3-thiazole scaffolds were documented to contribute to a variety of
antineoplastic potentials being employed as anticancer,9-13 anti-angiogenic,14 antiproliferative,15,16
tubulin polymerization inhibiting,17 and cytotoxic18,19 agents. Recently, it was reported that the
chemotherapeutic activity of 1,3-thiazoles derivatives are augmented by the discovery of
tiazofurin (natural antineoplastic antibiotic) (Fig 1, A),20 and S3U937 (Fig 1, B),21 which
exhibited potential antitumor activity against various cancer types.22 The documented DNA minor
groove binding property of 1,3-thiazole-netropsin, and thiazotropsin A (Fig 1, C and D) are also
payed attention.23,24 Another 1,3-thiazole derivative dasatinib (Fig 1, E) was reported to possess
potential tyrosine kinase inhibitory activity, and proved to be efficient in the treatment of imatinib
resistant mutants.25 Most of antitumor agents in Fig 1 are distinguished by containing substituted
1,3-thiazole ring, and carboxamide groups. Furthermore, recently literatures revealed that the
hydrazide-hydrazone (-CO-NH-N=CH-) moiety as a pharmacophore played a significant
antitumor activity in some antitumor agents.26-29 We noticed that some reported pharmacologically
active 1,3-thiazole derivatives exhibited a narrow activity spectrum,23,30 and their preparation
method is not so effective, and easily due to lower yields, long reaction time or under microwave
irradiation.31 It seemed to be reasonable to improve the synthetic method, and find new
1,3-thiazole derivatives with broad-spectrum antitumor activity.
In view of the above mentioned facts, we report herein the synthesis, in vitro cytotoxic
evaluation of some novel 1,3-thiazole derivatives based on pharmacophores hybridization. The
corresponding carboxamide moiety, and hydrazide-hydrazone pharmacophore were integrated

with structural unit of 1,3-thiazole into the novel structure T as shown in Fig 2, it was thought that
these novel skeleton T bearing three pharmacological groups would be well synergistic antitumor
effects, and broad-spectrum antitumor activity. Therefore
a
series of novel
2-(substituted(N-(methylcarbamoyl)
benzamide))-4-substituted-5-(substituted(N-benzylideneacetohydrazide))1,3-thiazole derivatives T
were designed and synthesized. It was expected that broad-spectrum antitumor activity and higher
synergistic effect could be achieved by chemical modifications of R1, R2, and R3 at the skeleton of
2,4,5-trisubstituted 1,3-thiazole derivatives T. We are here to report the synthesis, and evaluated
for their potential cytotoxic effects against human cancer cell lines of T. The possible
mechanism of antitumor activity for the highly potential compounds were also examined and
discussed.
Fig 1. Antitumor agents containing 1,3-thiazole ring and carboxamide moiety.
Fig 2. Design of T as antitumor agents.

The synthetic route of title compounds T is shown in Scheme 1.
Scheme 1. Synthesis of title compounds T1-46
The key skeleton of 1,3-thiazole ring was constructed by preparing ethyl 4-methyl(or 4-trifluoro
methyl)-2-(methylamino)1,3-thiazole-5-carboxylate M1 which is a key intermediate for the
synthesis of title compounds T. The preparation of M1 has been reported by several literatures.
31,32 However these methods are not effective, and need solvent, long reaction time or under
microwave irradiation. In our study, we could prepare M1a and M1b with 98% and 90% yield just
in 20 minutes under solvent-free condition. 4-Methyl(or trifluoromethyl)-2-methylamino-1,3-
thiazole-5-carbohydrazide M2 was prepared by hydrazinolysis of M1 using hydrazine hydrate
according to the method in literatures.33-37 Then M2 further reacted with substituted benzaldehyde
via the nucleophilic addition-eliminate reaction to produce key intermediate M3, constructing the
basic skeleton of 1,3-thiazole containing hydrazide-hydrazone moiety. The intermediate M6 could

be easily obtained according to the method in literatures.33-37 Subsequently, intermediate M3
reacted with substituted benzoylisocyanates M6 by the nucleophilic addition-eliminate reaction to
produce title compounds T1-46.
In order to find novel 2,4,5-trisubstituted 1,3-thiazole derivatives T with higher antitumor
activity, various substituents, especially, fluoro groups as R1, R2, and R3 were introduced into the
parent structure of M1, M3, and title compounds T to examine their effect on antitumor activity
against human breast cancer (MCF-7), hepatocellular liver carcinoma (HepG2), gastric cancer
(BGC-823), cervical carcinoma (Hela), and lung cancer (A549) cell lines by MTT method.38
5-Fluorouracil (5-Fu) was chosen as positive control due to its availability, and widespread using.
The inhibitory potency against five human cancer cell lines is showed in Fig 3. On the basis of
preliminary bioassay, the IC50 values of T1-38, M1a-b and M3a-h against five human cancer cell
lines were further tested to compare the effect of R1, R2, and R3 on cytotoxicity. The structure and
cytotoxicity (IC50) of T1-38 and M3a-h are listed in Table 1.
As shown in Fig 3A, M1 with the skeleton of 2-methylamino-1,3-thiazole-5-carboxylate only
showed <10% inhibitory potency against all tested cell lines. Although the cytotoxicity activity
could be enhanced by intruding hydrazide-hydrazone moiety into the skeleton of 1,3-thiazole M1
to produce M3, however M3 still showed <30% inhibitory potency. It was found that all title
compounds T except T3 exhibited much higher inhibitory potency against five human cancer cell
lines than that of corresponding M3 by the combination of 1,3-thiazole, hydrazide-hydrazone, and
carboxamide pharmacophore. Most title compounds T showed noticeable inhibitory activity
against BCG-823, and better than 5-Fu. T38 exhibited best selectively cytotoxic activity with 90%
inhibitory potency against HepG2 and its activity higher than 5-Fu. Above results indicated that

the combination of three pharmacophore including 1,3-thiazole, hydrazide-hydrazone, and
carboxamide was much favorable to cytotoxicity activity.
Fig 3. Inhibitory potency (%) of T1~38, M1 and M3 against five human cancer cell lines at 20μg/mL.
The data in Table 1 showed that all T1-38 exhibited moderate to excellent cytotoxic activity
against five tested human cancer cell lines with the IC50 values ranging from 1.11 to 65.46 μg/mL.
All the title compounds T, which combining of 1,3-thiazole, hydrazide-hydrazone, and
carboxamide pharmacophore, exhibited much higher inhibitory potency against five human cancer
cell lines than that of 1,3-thiazole1 M1, and 1,3-thiazole containing hydrazide-hydrazone moiety
M3, except T3 and T24 . Most of T displayed better or comparable inhibitory activity to 5-Fu
among thirty-eight tested compounds. It was found that eighteen compounds including T1, T2,
T4, T6~8, T10~12, T16, T19, T20, T26~30 and T38 displayed higher inhibitory activity against
all five human cancer cell than that of 5-Fu. Moreover, nine compounds including T5, T9, T16,
T17, T21~23, T25 and T36 exhibited better inhibitory activity against two or four human cancer

cell than that of 5-Fu. The results confirmed our hypothesis: incorporation of the
hydrazide-hydrazone, and carboxamide moiety into 1,3-thiazole scaffold could extend the
antitumor activity spectrum compared with these reported 1,3-thiazole.30
Table 1. Structure and cytotoxic activity of T1-38 and M3 against various cell lines
Substituents
R2
In vitro cytotoxicity IC50 (μg/mL)a
R1
R3
MCF-7b
HeLab
Hep-G2b
3.64±0.26
7.95±0.61
>100
BCG-823b A549b
HL-7702b
12.43±3.09
17.99±0.95
NTd
Compd
T1
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
4-CF3 2.21±0.9
4-CF3 8.95±1.67
4-CF3 >100
3.76±0.17
6.2±0.76
>100
2.53±0.82
1.84±0.44
>100
6.68±0.42
6.66±0.58
>100
T2
T3
CF3 3,5-(CF3)2 4-CF3 7.37±1.67
5.78±1.47
7.37±0.41
4.81±0.75
7.35±1.36
5.34±1.62
9.24±1.28
4.21±0.70
5.10±1.16
4.45±0.62
8.35±1.52
1.77±0.1
5.90±0.53
12.09+1.44
8.16±0.65
6.20±0.26
8.20±0.78
6.96±0.34
6.52±1.01
6.07±0.75
5.47±1.07
NTd
NTd
NTd
NTd
NTd
NTd
T4
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
3- CF3 7.69±1.72
3- CF3 3.70±0.96
3- CF3 6.50±1.51
10.41±0.38 7.29±2.31
T5
5.79±1.23
7.71±0.87
5.38±0.88
4.60±1.61
4.22±0.66
5.23±1.96
T6
T7
CF3 3,5-(CF3)2 3- CF3 6.08±0.29
T8
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
3,4-F2 8.21±1.64
3,4-F2 4.15±0.9
3,4-F2 5.93±1.11
10.38+2.11 3.66±0.47
T9
5.60±0.67
6.26±0.76
5.47±1.03
4.46±0.63
3.85±0.70
3.51±0.91
23.47±1.01
13.24±1.66
20.06±0.39
NTd
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
T26
T27
T28
T29
CF3 3,5-(CF3)2 3,4-F2 2.61±0.6
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
2,6-F2 20.43±1.28 19.56±1.98 26.09±4.51 16.95±0.92 17.88+2.88
2,6-F2 21.78±5.92 21.59±2.01 27.87+6.91 25.86±0.93 27.58+1.68
2,6-F2 35.33±6.29 30.59±3.12 49.59±6.35 34.88±6.83 40.12+3.56
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
NTd
CF3 3,5-(CF3)2 2,6-F2 6.85±1.12
5.62±1.02
6.28±2.37
5.28±0.52
4.67±1.12
17.3+1.79
6.32±1.23
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
2-F
2-F
2-F
2-F
12.23±1.44 10.12±0.98 5.39±1.56
20.54±5.73 19.12±1.32 20.34±2.42 16.34±1.45 19.45+2.32
4.98±1.1
3.97±0.98
4.98±1.11
9.00±0.46
9.6±1.62
7.99±0.66
>100
4.56±0.62
4.47±1.23
3.87±1.02
4.23±0.78
5.68±2.31
5.43±1.09
15.31+3.69
10.55+2.56
9.40±0.82
>100
CF3 3,5-(CF3)2
6.16±0.46
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
2,4-Cl2 9.12±2.69
2,4-Cl2 10.7±2.22
2,4-Cl2 8.42±2.12
10.65+0.86 5.73±1.91
11.18±1.03 6.11±1.65
12.41±0.56 3.08±0.54
CF3 3,5-(CF3)2 2,4-Cl2 >100
>100
>100
CF3
CF3
CF3
CF3
CF3
2- CF3
3- CF3
2- CF3
4-NO2
4-NO2
2,6-Cl2 6.24±3.1
4-NO2 4.39±0.16
3-NO2 4.73±0.93
3,4-F2 4.81±0.69
2,4-Cl2 6.74±1.20
5.23±0.34
4.12±0.65
4.15±0.76
5.08±0.22
5.12±0.78
8.95±1.66
4.73±0.91
4.80±1.43
6.23±1.45
6.85±1.70
4.12±0.65
1.67±0.43
3.97±0.61
2.99±0.45
2.13±0.45
12.00+1.21
5.42±0.53
5.45±0.89
5.78±0.87
6.44±1.09

CF3
CF3
Me
Me
3-NO2
3-NO2
3- CF3
4- CF3
2,4-Cl2 7.51±0.38
15.4±3.37
6.56±0.45
7.98±0.52
4.87±0.79
13.6±1.66
6.34±0.78
NTd
NTd
NTd
T30
T31
H
11.23±1.56 14.3±3.57
20.67+2.12
2,6-F2 28.15±5.21 26.78±2.11 31.57±5.17 32.65±3.12 30.32±1.89
2,6-F2 30.57±6.67 40.55±6.21 >100 65.46±7.35 60.45+5.33
T32
NTd
T33
Me 3,5-(CF3)2 2,6-F2 11.63±2.61 12.21±1.11 11.36±1.50 14.08±1.60 12.98+1.01
NTd
T34
Me
CF3
CF3
Me
Me
Me
Me
4- CF3
4-NO2
4-NO2
2- CF3
2- CF3
3- CF3
4- CF3
2,4-Cl2 24.52±3.57 20.32±2.34 >100
4-NO2 9.24±1.87 8.42±1.12 12.34_1.70 7.45±0.98
2-NO2 21.23±9.55 18.45±2.32 25.24±2.37 15.67±2.43 17.65+3.89
37.99±8.84 30.21+2.12
NTd
NTd
NTd
T35
8.98±1.23
T36
T37
2,6-F2 7.34±2.00
6.51±1.23
>100
1.11±0.26
>100
7.88±2.74
>100
5.43±1.56
>100
9.43±0.74
NTd
T38
-
-
-
-
-
-
-
-
-
>100
>100
M3-a
M3-b
M3-c
M3-d
M3-e
M3-f
M3-g
M3-h
5-Fuc
>100
>100
>100
>100
NTd
NTd
NTd
NTd
NTd
NTd
NTd
>100
>100
>100
>100
>100
Me 3,5-(CF3)2
>100
>100
>100
>100
>100
CF3
CF3
CF3
2- CF3
3- CF3
4- CF3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CF3 3,5-(CF3)2
>100
>100
>100
>100
>100
-
-
10.8±1.02
10.72±1.79 9.23±0.67
10.89±0.93 10.5±0.78
14.53±1.97
aIC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control
b
(72 h after treatment). MCF-7: human breast cancer cell lines; HepG2: hepatocellular liver carcinoma cell lines;
BGC-823: gastric cancer cell lines; Hela: cervical carcinoma cell lines; A549:lung cancer cell lines; HL-7702:
Human normal liver cell lines. c5-Fu: 5-Fluorouracil, used as a positive control. dNT: Not Test.
As shown in Table 1. The cytotoxic activity could be remarkably enhanced by optimizing the
structure of R1, R2, and R3. On the basis of 2,4,5-trisubstituted 1,3-thiazole ring, the introduction
of a trifluoromethyl group as R1 has a favourable effect on cytotoxic activity. Such as T14~16 and
T23 with CF3 as R1 exhibited higher cytotoxic activity than that of corresponding compounds
T32~35 with CH3 as R1. However, when 2-CF3 as R2, and 2,6-F2 as R3 were kept same, T38 with
Me as R1 had much higher cytotoxic activity than T13 with CF3 as R1. In addition T38 showed
higher cytotoxic activity against all five human cancer cells than that of 5-Fu and it was found to
be the most potent compound with specificity against HepG2 (IC50=1.11 μg/mL). These results

showed that the antitumor activity of T was not only depends on R1, but also depends on R2, and
R3. The structures of R3, and R2 are critical for antitumor activity.
The screening results also testified that the antitumor activity was highly dependent upon the
structure and position of substituent R2. T38 was found to be the most potent compound with IC50
values 1.11 μg/mL against HepG2. Thus the structure of T38 was chosen for further modification
to explore the effect of R2 on cytotoxic activity against HepG2. On the basis of the structure of
T38, Me as R1, 2,6-F2 as R3 were kept same, H, 2-Cl, 4-Cl, 2-F, 4-F, 2,4-Cl2, 3,4,5-(OCH3)3 or
4-OCH3 as R2 were introduced. The results were listed in Table 2. It was found that the cytotoxic
activity of compounds (such as T39~46) against HepG2 was very weak or almost lost
(IC50=22.66~70.1 μg/mL or > 100 μg/mL) when 2-CF3 as R2 was changed. This indicated that
2-CF3 as R2 was beneficial for antitumor activity. Hence, para, ortho, meta trifluoromethyl or
3,5-ditrifluoromethyl as R2 was kept, further modification was focused on the substituents of R3.
Table 2. Cytotoxic activity of T38-46 against HepG2
Compd.
Substituents
R2
In vitro cytotoxicity
IC50 (μg/mL)a
R1
R3
Me
Me
Me
Me
Me
Me
Me
Me
Me
2- CF3
H
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
1.1±0.26
T38
T39
T40
T41
T42
T43
T44
T45
T46
5-Fub
54.28
>100
2-Cl
4-Cl
2-F
>100
>100
4-F
>100
2,4-Cl2
70.01
>100
3,4,5-(OCH3)3
4-OCH3
22.66
9.23±0.67
aIC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control

(72 h after treatment). b5-Fu: 5-Fluorouracil, used as a positive control
SAR analyses showed that substituent R3 played a very important role in the antitumor activity
of T. When CF3 as R1 was kept, the substitutions of 3-CF3, 4-CF3 (except 4-CF3 as R2), 2-F
(except 2-CF3 or 3-CF3 as R2) or 3,4-F2 as R3 was very promotive for antitumor activity. For
example, T1, T2, T4, T6~8, T10~12, T19 and T20 displayed higher antitumor activity against all
five human cancer cell than that of positive control 5-Fu. Whereas, when CF3 as R1, the
introduction of 2,6-F2 as R3, such as T13~15 led to a sharp decrease in antitumor activity,
irrespective of ortho, para, or meta trifluoromethyl as R2, respectively. We noticed that when CF3
as R1 compound with 2,6-F2 as R3, 3,5-(CF3)2 as R2 or 2,6-Cl2 as R3, 2-CF3 as R2 the antitumor
activity was effectively increased, such as, T16 and T25 showed better inhibition than 5-Fu
against the four cell lines except A549 and much better than that of T13, T14 and T15. Above
observation indicated that the cytotoxic activity also depends on reasonable combination of R2 and
R3.
These interesting results indicated that improvement of antitumor activity required a reasonable
combination of R1, R2, and R3. T1 (R1=CF3, R2=2-CF3, R3=4-CF3), T26 (R1=CF3, R2=3-CF3, R3=
4-NO2) and T38 (R1=Me, R2=2-CF3, R3=2,6-F2) were found to be the most promising compounds
with wide spectrum, and good activity against five tested human cancer cell lines (IC50=1.11~7.88
μg/mL), their activity were much better than that of positive control 5-Fu (IC50=9.23~10.89
μg/mL). They might be used to develop novel lead scaffolds for potential anticancer agents. In
addition, some activity compounds against cancer cell lines were selected for testing their toxicity
against human normal liver cell (HL-7702). The data in Table 1 showed that T10 (IC50=23.47

μg/mL) and T12 (IC50=20.06 μg/mL) showed weak toxicity on normal cell line compared with
5-Fu (IC50=14.53 μg/mL).
To determine whether the observed cell death induced by 1,3-thiazole derivatives (T) was due
to apoptosis or necrosis, the interaction of HepG2 cells with T1 and T38 was further investigated
using an AnnexinV-FITC/PI (Propidium iodide) dual staining assay39. All data obtained in this
study are presented in Fig 4, as a percentages of intact cells at lower-left quadrant,
FITC(－)/PI(－); early apoptotic cells at lower-right quadrant, FITC(+)/PI(－); late apoptotic or
necrotic cells at upper-right quadrant, FITC(+)/PI(+); necrotic cells at upper-left quadrant,
FITC(-)/PI(+). The apoptotic cell rates were determined for HepG2 cells that were treated with T1
and T38 at the concentration of 1.82 μg/mL (0.5IC50 value), 3.64 μg/mL (IC50 value) and 0. 55
μg/mL (0.5IC50 value), IC50 1.1 μg/mL (IC50 value) for 24h, respectively. It can be observed from
Fig 4F that T1 and T38 significantly caused the early apoptosis in terms of FITC(+)PI(-) staining.
T1 displayed 7.8% and 18.6% of apoptosis at 0.5 IC50 and IC50 concentrations, respectively. T38
displayed 7.0% and 16.5% of apoptosis at 0.5 IC50 and IC50 concentrations, respectively. However,
the control (0.1% DMSO) displayed 2.5% of apoptosis. The results showed that T1 and T38
induced cell death in HepG2 cells at least partly (initially) by apoptosis.

A Control
B T1 0.5IC50
C T1 IC50
D T38 0.5IC50
E T38 IC50
F 24h Treatment
Early apoptotic cells
Fig 4. Flow cytometric analysis of HepG2 cells treated with synthetic compounds T1 and T38. Cells were stained
with Annexin V-FITC/PI and quantitated by flow cytometry. A) Not treated with compound, as control; B) Treated
with T1 at 1.82 μg/mL (0.5IC50 value); C) Treated with T1 at 3.64 μg/mL (IC50 value); D) Treated with T38 at 0.
55 μg/mL (0.5IC50 value) ; E) Treated with T38 at 1.1 μg/mL ( IC50 value) for 24h; F) Apoptotic effect of
compounds T1 and T38 was evaluated after 24 h treatment; bar graphs represent mean ± SD in at least three
independent experiments.
The cell cycle is the series of proceedings that occur in a cell resulting in its division and
duplication. The cell cycle involves four distinct phases: G1 phase, S phase (synthesis), G2 phase
(collectively known as interphase), and M phase (mitosis). The G1 phase is the preparation of
energy, and material for DNA replication. The S phase is DNA replication. The G2 phase is the
preparation for the M phase. The M phase is “mitosis”, and is nuclear and cytoplasmic division.
To determine whether the suppression of cancer cell growth using T38 was caused by a cell cycle

progression, a cell-cycle cytotoxicity assay was performed by treating HepG2 cells with T38 at
the concentration of 0. 55 μg/mL (0.5IC50 value) and 1.1μg/mL (IC50 value) for 24h respectively.
Antitumor activity of T38 can be accompanied by cell cycle arrest in any of the four phases
(sub-G1, G1, S, and G2). As shown in Fig 5, cells in the G1 phase decreased from 57.68% as
control to 41.02% and 11.83% respectively. A decrease of population in G2 phase from 6.89% as
control to 2.11% and 0 respectively was also found when the concentration was increased to
0.5IC50 and IC50. As control, the cell population in the S phase of the cell cycle was 35.42%,
which could be increased to 56.87% at the concentration of 0.5IC50 and 88.17% at the
concentration of IC50 respectively. Meanwhile, as obviously increase of cell number in the sub-G1
phase was observed from 0 to 34.48% and 66.72% after treatment with T38 at the concentration of
0.5IC50 value and IC50 value. Hence, T38 induced a markedly S phase arrest in a concentration
manner and subsequent cell death.

D
Fig 5. Cell-cycle analysis of T38 by flow cytometry in HepG2 cells. A) Not treated with T38 as control at for 48h;
B, C) treatment with T38 at 0.55 μg/mL (0.5IC50 value) and 1.1 μg·mL (IC50 value) for 48h, respectively. D) The
numbers of sub-G1, G1, S, G2 phase cells were expressed as a percentage of the total cell number.
In summary, a series of novel 2,4,5-trisubstituted 1,3-thiazole derivatives containing
hydrazide-hydrazone, and carboxamide moiety including 46 compounds were designed and
synthesized. The SAR analyses indicated that the satisfactory cytotoxic activity required a
reasonable combination of R1, R2, and R3 in parent structure T. Antitumor activity could be
increased greatly by optimizing R2, and R3. Eighteen compounds displayed higher inhibitory
activity against all five human cancer cells than that of 5-Fu. T1, T26 and T38 exhibit best
cytotoxic activity with IC50 values of 2.21 μg/mL, 1.67 μg/mL and 1.11 μg/mL, against MCF-7,
BCG-823 and HepG2 cell lines, respectively. Especially, T1, T26 and T38 were found to be the

most promising compounds with wide spectrum and much higher activity (IC50=1.11~7.88 μg/mL)
than that of positive control 5-Fu (IC50=9.23~10.89 μg/mL) against all tested cell lines. The results
showed that we could obtain compounds with good antitumor activity and extend the activity
spectrum by the introduction of the hydrazide-hydrazone, and carboxamide moiety into
1,3-thiazole scaffold to form 2,4,5-trisubstituted 1,3-thiazole derivatives. In addition, further
studies revealed that T1 and T38 could induce apoptosis in HepG2 cells and it was confirmed T38
led the induction of cell apoptosis through S cell-cycle arrest. This suggested that the scaffold of
2,4,5-trisubstituted 1, 3-thiazole containing hydrazide-hydrazone and carboxamide moiety could
be used as a lead structure for further optimization to find more potent antitumor agents.
Acknowledgment
The work was supported in part by the National Basic Research Program of China (No.
2010CB126100); National Natural Science Foundation of China (Nos. 21172090 and 21472062);
Natural Science Key Foundation of Hubei Province (No. 2014CFA111); Excellent Doctorial
Dissertation Cultivation Grant From Central China Normal University.
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Synthesis, antitumor activity and mechanism of action of novel 1,3-thiazole
derivatives containing hydrazide-hydrazone and carboxamide moiety
Haifeng He,a Xiaoyan Wang,a Liqiao Shi,b Wenyan Yinb, Ziwen Yangb, Hongwu He,a,* and Ying
Liangb,*
Graphical Abstract
Most of design compounds T displayed significantly inhibition activityu against human breast
cancer (MCF-7), hepatocellular liver carcinoma (HepG2), gastric cancer (BGC-823), cervical
carcinoma (Hela) and lung cancer (A549) cell lines with the control 5-fluorouracil. Especially,
T38 could induce apoptosis in HepG2 cells by S cell-cycle arrest.